VAR2CSA Ectodomain Labeling in Plasmodium falciparum Infected Red Blood Cells and Analysis via Flow Cytometry

Presentation of the variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (EMP1) at the surface of infected red blood cells (RBCs) underpins the malaria parasite’s pathogenicity. The transport of EMP1 to the RBC surface is facilitated by a parasite-derived trafficking system, in which over 500 parasite proteins are exported into the host cell cytoplasm. To understand how genetic ablation of selected exported proteins affects EMP1 transport, several EMP1 surface presentation assays have been developed, including: 1) trypsinization of surface-exposed EMP1 and analysis by SDS-PAGE and immunoblotting; and 2) infected RBC binding assays, to determine binding efficiency to immobilized ligand under physiological flow conditions. Here, we describe a third EMP1 surface presentation assay, where antibodies to the ectodomain of EMP1 and flow cytometry are used to quantify surface-exposed EMP1 in live cells. The advantages of this assay include higher throughput capacity and data better suited for robust quantitative analysis. This protocol can also be applied to other cellular contexts where an antibody can be developed for the ectodomain of the protein of interest.


Background
The most virulent form of malaria is caused by the protozoan parasite Plasmodium falciparum, killing over 600,000 people annually (Weiss et al., 2019). During the asexual blood stage, the parasite invades the red blood cell (RBC) and feeds on intracellular hemoglobin; however, while circulating in the host bloodstream, the infected RBC is at risk of elimination if it transits the host's splenic sinuses. To avoid splenic clearance, the parasite remodels the host cell by exporting proteins into the RBC cytoplasm (Marti et al., 2004;Sargeant et al., 2006;Boddey et al., 2013;Heiber et al., 2013). During this process, a key modification is the presentation of the variant antigen P. falciparum erythrocyte membrane protein 1 (EMP1) at the surface of infected RBCs (Smith et al., 1995). The antigen EMP1 is encoded by the var gene family where each parasite expressing one of approximately 60 variants at any time. These variants act as adhesins to a range of cellular ligands, expressed on a range of endothelial cells in the capillaries throughout the body, allowing the infected RBC to sequester within the host's vasculature. The cytoadhesion of infected RBCs can lead to fatal complications associated with cerebral and placental malaria (Storm and Craig, 2014;Jensen et al., 2020;Sahu et al., 2021). EMP1 transport to the surface remains poorly understood as the parasite, with the parasite building its own de novo trafficking system, which is highly divergent from classical eukaryotic trafficking machinery. For these reasons, EMP1 trafficking is of high interest from both clinical and basic biology perspectives. In our recent studies, we identified parasite proteins that are exported into the host cell and affect the trafficking and presentation of the antigen EMP1 (McHugh et al., 2020; Carmo et al., 2022). The majority of EMP1 is trafficked to the host cell surface 16-20 h post invasion, and mid-trophozoite stage-infected RBCs (20-32 h post invasion) are used to study EMP1 presentation at the host cell surface (Kriek et al., 2003). To determine if EMP1 is present at the surface of infected RBCs, we employ two complementary assays. These include: 1) trypsin cleavage assay, where surface-exposed EMP1 is shaved from the surface by trypsin and the membrane-embedded domain is then detected by immunoblotting ( . These assays are useful; however, they have their limitations. For example, the trypsin assay is semi-quantitative at best, while defects in adhesion underflow can be multifactorial (not due to EMP1 surface presentation alone). In an alternative approach outlined here, antibodies specific to the ectodomain of EMP1 are used to label live cells, which are then quantitated by flow cytometry, to measure the number of labeled cells and their relative intensity of surface-exposed EMP1 (first established in Smith et al., 1995;Beeson et al., 2004;Elliott et al., 2005) (Figure 1). Of the techniques assaying EMP1 surface presentation published to date, this is the most time-and resource-efficient method. In addition, the assay is implemented in a 96-well plate format, making it easily scalable. This technique can also be adjusted to quantitate surface presentation of surface proteins in other cellular contexts where an antibody is available for the ectodomain of the protein of interest.

Procedure
All sample preparation is performed under sterile conditions. Centrifugation steps are at either 528× g for 5 min (5 acc/1 dec) (A3-B1) or 528× g for 90 s (9 acc/9 dec) for 96-well plates. Slow deceleration is used when centrifugation is performed in Falcon tubes to reduce the disturbance of the infected RBCs pellet.
A. Prepare malaria parasite cell culture 1. Maintain parasite-infected RBC culture at 5% hematocrit (percentage by volume of RBCs in culture) with O+ RBCs in CCM at 37 °C in a low oxygen environment (malaria mix). As an example, a 35 mm × 10 mm tissue culture dish would contain 250 μL of RBCs and 4.75 mL of CCM, yielding a 5% hematocrit culture. O+ RBCs are used as they are compatible with pooled sera from any blood type, avoiding RBC agglutination. 2. Parasitemia (the percentage of infected RBCs relative to total RBCs) should be kept at ≤ 5% and the CCM replaced every 24-48 h. Monitor the parasitemia via thin blood smears and Giemsa staining. a. Thin blood smears and Giemsa staining: Deposit 1-2 μL of RBCs at one end of a glass microscope slide. Use another glass slide held at a 45° angle to smear the cells across the first slide. Allow to air dry; then, fix the cells by immersing the slide in methanol for at least 5 s. Allow the slide to air dry and incubate the fixed slide in 10% Giemsa stain diluted in water for 5 min. Remove the slide from the stain and wash off excess stain with tap water. Blot the slide dry with paper towels; then, add immersion oil and visualize the cells with a 100× objective on a light microscope. Cell fixation and staining can be done in a non-sterile environment. Parasites will be stained dark purple and RBCs light purple. Use a physical cell counter to count ≥ 500 cells to determine the parasitemia, or percentage of cells infected with a parasite, prior to an experiment. Harvest the culture by centrifugation at 528× g for 5 min (5 acc/1 dec), aspirate the media, and resuspend the pellet in 5% D-sorbitol (w/v) to obtain a 5% hematocrit solution (the original volume of the culture). Incubate the D-sorbitol-treated culture in a water bath at 37 °C for 5-10 min. Harvest the sorbitol-treated culture, aspirate the supernatant, resuspend the pellet in CCM to 5% hematocrit, and return the culture to a fresh culturing dish or flask. One sorbitol synchronization will retain parasites that are ~0-20 hpi. Sorbitol-synchronize the parasites again 8 h later to narrow the age range to ~8-20 hpi. For the protocol outlined here, parasites must be synchronized to ~20-32 hpi (i.e., two synchronizations, 12 h apart).  1. The infected RBC culture should be at 5% hematocrit with a parasitemia between 3% and 5%. Harvest mid-trophozoite stage cultures (~20-32 hpi) by centrifugation at 528× g for 5 min (5 acc/1 dec). 2. Aspirate the spent media leaving the RBC pellet undisturbed. Resuspend the RBCs in 1% BSA/PBS and dilute as required to obtain a solution containing a 3%-5% parasitemia and 2%-4% hematocrit. 3. Load 20 μL of diluted cells into a 96-well plate in duplicate or triplicate per condition and cell line. At a minimum, you will require a no-primary control (wells incubated with the BSA diluent and that receive the second and third antibody treatments), totalling ≥ 6 wells per cell line if performed in triplicate. 4. Add 100 μL of wash buffer (1% BSA/PBS) to each well. Alternatively, add 100 µL of wash buffer prior to loading cells in step B3. We recommend a multichannel pipette for all subsequent washing steps. 5. Centrifuge the plate at 528× g for 90 s (9 acc/9 dec) to pellet the cells, then aspirate the media using an aspirating tip. We find that the accuracy of aspiration is improved when a sterile pipette tip (no filter) is mounted on the end of the aspirator tip. Touch the tip to the wall of each well to aspirate the supernatant.

C. Cell staining
Note: All antisera are diluted in wash buffer (1% BSA/PBS).

D. Flow cytometry using FACSDiva software
The steps below refer to a non-GFP-expressing cell line, labeled with Alexa Fluor 488 tertiary antibody and Hoechst 33342. Perform these steps for one well containing a double-stained positive sample (or well), e.g., the positive control, then apply the gating strategy to all wells in the plate. 1. Use a side scatter height (SSC-H) vs. forward scatter area (FSC-A) plot to identify and gate the total RBC population (Figure 2A). Select this population. 2. Then, use a forward scatter height (FSC-H) vs. forward scatter width (FSC-W) plot to gate singlet events ( Figure 2B). Select this population. 3. Plot Pacific blue height (DNA stain) vs. FITC height (fluorophore-conjugated tertiary antibody) ( Figure  2C). Adjust the voltage of Pacific blue and FITC so that the Pacific blue-H +/-and FITC-H +/-populations can be clearly delineated. In our example plot ( Figure 2C), the quadrant divide sits at approximately 10 3 .
Once the voltages are optimized, apply these acquisition paraments to all wells in the plate and all biological repeats. As visualized in Figure 1, the Q4 population represents uninfected RBCs, the Q1 population is infected but does not have labeled EMP1 on the surface, and the Q2 population is both infected and presenting EMP1 on the surface. 5. Collect a total of 50,000 events per well.